MOLECULAR MEDICINE REPORTS 13: 5013-5020, 2016
Ketamine attenuates osteoarthritis of the knee via modulation of inflammatory responses in a rabbit model WEI LU1, LIN WANG1, CHUNXIN WO1,2 and JING YAO1 1
Department of Anesthesiology, Guizhou Medical University; 2Division of Pain Management, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550004, P.R. China Received May 28, 2015; Accepted March 31, 2016 DOI: 10.3892/mmr.2016.5164 Abstract. The aim of the present study was to investigate the efficacy of ketamine in attenuating osteoarthritis (OA) and modulating the expression of inflammatory mediators. A rabbit OA model was established by knee immobilization using plaster bandages. After six weeks, rabbits were randomly allocated into four groups (n=6/group): Normal saline, Ket60, Ket100, and Ket200 and twice a week for four weeks the rabbits received an intra‑articular injection of saline, or 60, 100 or 200 µmol/l ketamine, respectively. One week after the final injection, samples of synovial membrane, synovial fluid and articular cartilage were isolated. The pathological changes were assessed by general observation, hematoxylin and eosin staining and Alcian blue/periodic‑acid Schiff staining. Cartilage pathology was assessed using Mankin's scoring system. Tumor necrosis factor (TNF)‑α and interleukin (IL)‑10 levels in the synovial fluid were measured by enzyme‑linked immunosorbent assays. The nuclear factor (NF)‑ κ B p65 subunit expression level in cartilage samples was determined by immunohistochemistry. OA was characterized by morphological changes in the articular surface, cartilage lesions, infiltration of inflammatory cells and a significantly increased Mankin's score. Elevated TNF‑α and reduced IL‑10 levels in the synovial fluid, along with increased p65 expression levels in the cartilage were observed in OA rabbits. Intra‑articular injection of ketamine ameliorated the pathological characteristics of OA, reduced the Mankin's score, decreased TNF‑α and NF‑κ B p65 expression levels, and increased the level of IL‑10 expression in a dose‑dependent manner. Thus is was demonstrated that Ketamine suppresses the inflammatory response in OA by modulating inflammatory mediator expression levels in a rabbit model of OA.
Correspondence
to: Ms. Chunxin Wo, Department of Anesthesiology, Guizhou Medical University, 4 Beijing Road, Guiyang, Guizhou 550004, P.R. China E‑mail:
[email protected] Key words: ketamine, osteoarthritis, tumor necrosis factor‑α, interleukin‑10, nuclear factor‑κ B
Introduction Osteoarthritis (OA) is the most common form of progressive joint disease in the elderly, which has a reported prevalence of 34.18% in women and 30.18% in men, aged 65 years or older, in the United States of America (1). A previous study conducted in Beijing, China, revealed a higher prevalence of OA in Chinese women of a similar age (46.16%) and a comparable prevalence in Chinese men (27.16%) (2). OA is associated with degenerative changes in the joints, along with joint diseases caused by loss of the cartilage matrix (3). Furthermore, previous studies have highlighted the role of inflammation in the pathogenesis of OA (4,5). Elevated expression levels of pro‑inflammatory cytokines and mediators, including interleukin (IL)‑1, IL‑6, tumor necrosis factor (TNF)‑ α and nitric oxide (NO) are present in the serum and synovial fluid of OA patients (6,7). Anti‑inflammatory agents, such as IL‑1β inhibitor, diacerein and TNF‑ α inhibitors, infliximab and etanercept have demonstrated promising clinical efficacy for the treatment of OA (8‑10). However, to the best of our knowledge, there have been no reports of therapeutic strategies that could simultaneously engage multiple inflammatory mediators and alleviate inflammation‑induced tissue damage. Recent studies have extended the functional scope of glutamate beyond neuronal tissues to a wide range of tissue and cell types, exhibiting an autocrine or paracrine effect (11‑13). McNearney et al (14) revealed increased levels of glutamate, and pro‑inflammatory cytokines and chemokines in the synovial fluid of patients with active arthritis. Glutamate interacts specifically with the N‑methyl‑D‑aspartate (NMDA) receptor and activates the downstream signaling cascade (15). Flood et al (16) proposed the role of NMDA receptor in inflammatory joint damage in rheumatoid arthritis. In an animal study, injecting glutamate into the knee joint of rats led to thermal hyperalgesia and mechanical allodynia, which was alleviated by subsequent injections of NMDA receptor antagonists (17). Magnesium is an NMDA receptor antagonist, and an articular injection has been shown to improve the degree of pain following arthroscopic surgery and improve experimental rat bone arthritis (18,19). Results from the above‑mentioned studies indicate the therapeutic potential of glutamate/NMDA receptor signaling in OA. Ketamine is a non‑competitive antagonist of the NMDA receptor, and has been widely administered as an anesthetic and pain killer (20,21). Recent studies
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indicate that ketamine exhibited potent anti‑inflammatory activity in various inflammatory disease models when administered at or below clinical dosages. Ketamine antagonizes the NMDA receptor to regulate calcium influx, increases the intracellular concentration of cyclic adenosine monophosphate, inhibits the formation of oxygen radicals and inducible NO synthase (iNOS) following polymorphonuclear activation, and modulates the production of various pro‑inflammatory mediators (21). Mechanistic studies indicate that ketamine directly inhibits the expression of nuclear factor (NF)‑κ B, which is a master regulator of pro‑inflammatory cytokine transcriptions (22). In addition, ketamine exhibits protective effects against oxidative stress by inhibiting iNOS activity and decreasing nitrite/nitrate levels (23). These findings highlight the therapeutic potential of ketamine in inflammatory disorders, including OA. In the present study, a rabbit OA model was established by immobilizing the knee joint, as previously described (24), and the efficacy of different doses of ketamine in ameliorating the inflammatory response was evaluated. In addition, the current study investigated the anti‑inflammatory role of ketamine in regulating multiple inflammatory cytokines and signaling molecules for possible therapeutic application in OA. Materials and methods Animals and reagents. Thirty skeletally mature, male, New Zealand white rabbits (weight, 2.5‑3 kg) were obtained from the Experimental Animal Center of Guiyang Medical School (Guiyang, China) and were acclimated for one week prior to the experimental procedures. All animal procedures were approved by the Institutional Ethics Committee. The ketamine used in the present study was a 1:1 racemic mixture of two enantiomers, which was purchased from Yichang Humanwell Pharmaceutical Co., Ltd. (Yichang, China). Enzyme‑linked immunosorbent assay (ELISA) kits for IL‑10 and TNF‑α, and the anti‑NF‑κB p65 antibody were purchased from Shanghai Bogoo Biotechnology Co., Ltd. (Shanghai, China). The 3,3'‑diaminobenzidine (DAB) Substrate kit was obtained from ZSGB‑Bio Co., Ltd. (Beijing, China), and Alcian blue/periodic acid‑Schiff (AB/PAS) Stain kit was obtained from Huanyu Jinying Technology Co., Ltd. (Beijing, China). Rabbit OA model and ketamine treatment. Twenty‑four rabbits were randomly selected to establish the OA model. The left knee joint was immobilized (3 cm above the rear ankle to 1.5 cm below the groin) for six weeks using plaster bandages, with the knee flexed at 30‑40˚. Dorsalis pedis pulses were detected on each side. Plaster tightness was examined for three consecutive days after creating the model and were adjusted as appropriate. The rabbits were housed in separate cages and forced to move or exercise occasionally. Six rabbits were left untreated and served as a normal control. After six weeks, the plaster was removed and rabbits were randomly allocated into four groups: Normal saline, Ket60, Ket100 and Ket200. Saline or various concentrations of ketamine (60, 100 and 200 µmol/l) were injected into the articular cavity in 0.5‑ml volumes, twice a week for four weeks. One week after the last injection, sample collection was conducted, then all rabbits were sacrificed under intravenous anesthesia with
25% urethane (4 ml/kg; Shanghai-Rui Biological Technology Co., Ltd., Shanghai, China) by air injection (20 ml) into an ear marginal vein. Sample collection. The thigh was rotated and an incision was made on the inner side in order to peel back the skin. The quadriceps were dissected 0.5 cm above the knee joint, and separated from the femur and patella. The synovial membrane under the patella was removed using ophthalmic scissors (Shanghai Yuyan Instruments Co., Ltd., Shanghai, China) and rinsed in ice‑cold phosphate‑buffered saline solution (Shanghai-Rui Biological Technology Co., Ltd.), and fixed in 10% neutral buffered formalin (BioSynTech, Beijing, China) for 24 h followed by dehydration, clearing, wax infiltration and embedding in paraffin (Junruishengwu Technology Corporation, Shanghai, China) for further histological examination. The samples were 0.5 x 1 x 0.5 cm in size. The femoral condyle and a portion of the cartilage from the knee joint were removed, rinsed and immediately fixed in 10% neutral buffered formalin for 24 h. Samples were decalcified prior to histological examination. Synovial fluid was obtained from the knee joint by injecting 0.5 ml saline solution followed by aspiration, which was performed three times. Synovial fluid (~0.8‑1 ml) was extracted and samples were centrifuged at 2147 x g for 10 min. The supernatants were obtained and stored at ‑70˚C until further use. General and histopathological examination. The knee joint was observed for signs of synovitis, including joint effusion and swelling. Pathological changes in the articular surface of the medial femoral condyle were evaluated under an Olympus CX41 biological microscope (Olympus Corporation, Tokyo, Japan). Samples were scored according to the following criteria (25): 0, Smooth articular surface with normal color; i) rough articular surface with gray color and small cracks; ii) eroded articular surface with cartilage lesions penetrating the middle layer; iii) ulcers on articular surface with cartilage lesions penetrating the deep layer; and iv) complete loss of cartilage with exposed subchondral bone. Paraffin‑embedded tissue samples were sectioned into 3‑5‑µm thick slices using a DQP‑9010 rotary slicer (Shanghai Huayan Equipment Co., Ltd., Shanghai, China). The synovial membrane was stained with hematoxylin and eosin (H&E; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and mounted with neutral balata (Shanghai-Rui Biological Technology Co., Ltd.); the pathological changes were evaluated by two independent observers. The cartilage was stained using H&E and AB/PAS, and mounted with neutral balata. Samples were scored by two independent observers according to Mankin's score system (26) and the mean was taken as the final score. ELISA of IL‑10 and TNF‑ α in the synovial fluid. IL‑10 and TNF‑ α concentration levels in the synovial fluid were measured using specific ELISA kits according to the manufacturer's instructions. Absorbance (optical density value) at a wavelength of 450 nm was determined using a UV‑2600 microplate reader (Shimadzu Corporation, Kyoto, Japan). Im munohistochemical a nalysis of NF‑κB. Immunohistochemical expression of NF‑κ B in the cartilage
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was determined using the Labeled Streptavidin Biotin method. Samples were evaluated based on positive DAB staining in the cytoplasm and nucleus of chondrocytes: Weak positive, pale yellow stain; positive, yellow stain; medium positive, brown stain; strong positive, tan stain. Eight fields (magnification, x400) from each section were randomly selected and the positive rate (%) was determined by the percentage of positively‑stained cells. Results were assessed by an investigator and two experienced technicians. Statistical analysis. Data were expressed as arithmetic or geometric mean ± standard deviation. Means of the two groups were compared using Levene's test of homogeneity of variance. Homogeneous variance was analyzed by one‑way analysis of variance, and pairwise comparison between groups was made by Fisher's least significant difference test. Dunnett's T3 test was performed when there was lack of homogeneity of variances. Positive rates were compared by χ2‑test. P
